PROJECT SUMMARY Two opposing signaling pathways which are critical regulators of cell growth and metabolism are the AMPK and mTOR pathways. mTOR generally promotes cellular growth and proliferation, while AMPK inhibits mTOR and generally suppresses growth and proliferation. Both pathways are acutely responsive to cellular nutrient and energy levels and demands. An adenylate kinase (AK4) has been identified whose expression regulates cellular adenosine levels and concurrent activation of AMPK and mTOR, resulting in both enhanced proliferation and activation of responses such as autophagy in multiple cell types. These findings position AK4 upstream of both signaling pathways, with AK4 expression able to coordinate activation of both pathways. Each of these pathways are important for maintaining cellular homeostasis, and dysregulated mTOR and AMPK signaling are implicated in numerous human diseases. The objectives of this proposal are to characterize the mechanism by which AK4 regulates adenosine nucleotide pools, concurrent AMPK and mTOR signaling, and the downstream biological effects of AK4 expression-mediated mTOR and AMPK activation. The aims of this proposal are 1) to identify the mechanistic basis whereby AK4 exerts its biological effects; 2) to identify the mechanism whereby AK4 regulates concomitant AMPK and mTOR activation; 3) to define the functional consequences of AK4-mediated activation of mTOR- and AMPK-dependent biological actions on cell biology. To achieve these goals, AK4 expression levels will be genetically enhanced or suppressed, and relevant mechanisms which may modulate ATP levels and mTOR and AMPK signaling will be assessed. These include measuring glycolytic and OXPHOS flux, adenosine nucleotide biosynthesis, subcellular localization of activated AMPK and mTOR, and effector protein status. Finally, the biological effects (protein synthesis, proliferation, autophagy, mitochondrial biogenesis, etc.) of these cellular events will be characterized for their potential to contribute to cellular homeostasis. Together, these goals and approaches will define the mechanistic basis by which AK4 regulates AMPK and mTOR signaling.